WO2013043981A1 - Cellulolytic enzyme compositions and uses thereof - Google Patents

Cellulolytic enzyme compositions and uses thereof Download PDF

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Publication number
WO2013043981A1
WO2013043981A1 PCT/US2012/056502 US2012056502W WO2013043981A1 WO 2013043981 A1 WO2013043981 A1 WO 2013043981A1 US 2012056502 W US2012056502 W US 2012056502W WO 2013043981 A1 WO2013043981 A1 WO 2013043981A1
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Prior art keywords
seq
strain
cellulolytic
acetylxylan esterase
cellulosic material
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PCT/US2012/056502
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English (en)
French (fr)
Inventor
Prashant Iyer
Armindo Ribeiro GASPAR
James CROONENBERGHS
Thomas P. Binder
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Novozymes A/S
Archer Daniels Midland Company
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Application filed by Novozymes A/S, Archer Daniels Midland Company filed Critical Novozymes A/S
Priority to EP12768989.1A priority Critical patent/EP2748313B1/en
Priority to CA2849885A priority patent/CA2849885A1/en
Priority to CN201280057791.0A priority patent/CN104039958A/zh
Priority to BR112014006853A priority patent/BR112014006853A2/pt
Priority to US14/343,002 priority patent/US9714416B2/en
Priority to DK12768989.1T priority patent/DK2748313T3/da
Publication of WO2013043981A1 publication Critical patent/WO2013043981A1/en
Priority to US14/279,559 priority patent/US20140322766A1/en
Priority to US14/279,550 priority patent/US20140322763A1/en
Priority to US15/629,695 priority patent/US20170342391A1/en

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    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
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    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
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    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
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    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
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    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
    • C12P7/10—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate substrate containing cellulosic material
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    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • C12P7/14—Multiple stages of fermentation; Multiple types of microorganisms or re-use of microorganisms
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    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
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    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01021—Beta-glucosidase (3.2.1.21)
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    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
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    • C12P2203/00—Fermentation products obtained from optionally pretreated or hydrolyzed cellulosic or lignocellulosic material as the carbon source
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • the present invention relates to cellulolytic enzyme compositions; methods of using such cellulolytic composition for hydrolyzing acetylated cellulosic materials; and processes of producing fermentation products using cellulolytic enzyme compositions of the invention.
  • the glucose is easily fermented by yeast into ethanol.
  • the rate and extent of enzymatic hydrolysis of cellulosic material depends on various structural features such lignin content, acetyl content and crystallinity.
  • hemicellulosic material such as xylan
  • cellulosic material cellulose
  • acetylation occurs when cellulosic material is subjected to pretreatment with, e.g., acids, such as acetic acid.
  • acids such as acetic acid
  • WO 2005/047499 discloses an Aspergillus fumigatus beta-glucosidase and gene thereof.
  • WO 2006/078256 discloses Aspergillus fumigatus GH10 xylanases.
  • WO 2008/151079 discloses compositions for degrading cellulose material.
  • WO 2009/042846 disclosed an acetylxylan esterase (AXE) derived from Thielavia terrestris.
  • WO 201 1/041397 discloses a Penicillium sp. GH61 polypeptide having cellulolytic enhancing activity and gene thereof.
  • WO 201 1/057140 discloses an Aspergillus fumigatus cellobiohydrolase I; Aspergillus fumigatus cellobiohydrolase II; and an Aspergillus fumigatus beta-xylosidase.
  • the present invention relates to enzyme compositions comprising cellulolytic activity; the use thereof for hydrolyzing acetylated cellulosic materials; and processes of producing fermentation products using a cellulolytic enzyme composition of the invention.
  • the invention relates to an enzyme composition
  • a cellulolytic preparation and an acetylxylan esterase (AXE).
  • AXE acetylxylan esterase
  • the invention relates to methods of hydrolyzing acetylated cellulosic material, comprising subjecting the acetylated cellulosic material to an enzyme composition of the invention comprising a cellulolytic preparation and an acetylxylan esterase (AXE).
  • an enzyme composition of the invention comprising a cellulolytic preparation and an acetylxylan esterase (AXE).
  • the acetylxylan esterase is derived from a strain of the genus Thielavia, such as a strain of Thielavia terrestris, such as one disclosed in WO 2009/042846 as SEQ ID NO: 2 or SEQ ID NO: 1 herein or an acetylxylan esterase having at least 80%, such as at least 85%, such as at least 90%, preferably 95%, such as at least 96%, such as 97%, such as at least 98%, such as at least 99% identity to SEQ ID NO: 2 in WO 2009/042846 or SEQ ID NO: 1 herein.
  • the invention relates to processes of producing a fermentation product from acetylated cellulosic material, comprising:
  • Figure 1 shows the glucose yield (%) after 1 1 days hydrolysis.
  • Figure 2 shows the xylose yield (%) after 1 1 days hydrolysis.
  • Figure 3 shows the results from pretreated acetylated corn stover pulp (5% solids, pH 5.2, 50°C, 6 mg protein/g cellulose).
  • Acetylated Cellulosic material refers to cellulosic material that has a higher degree of acetylation than native cellulosic material.
  • the acetylation-% for the target cellulosic substrate may be as high as 30% (average acetyl groups per sugar units).
  • the acetylated cellulosic material is 0.1-30%, such as 0.5-20%, preferably 1-10%, such as around 5-10%, such as around 8% acetylated.
  • Acetylation can be determined according to the NREL procedures described in Technical Report NREL/TP-510-42618 (Revised July 201 1 ) and as described in the "Materials & Methods" section.
  • Cellulosic material means any material containing cellulose.
  • the predominant polysaccharide in the primary cell wall of cellulosic material is cellulose, the second most abundant is hemicellulose, and the third is pectin.
  • the secondary cell wall, produced after the cell has stopped growing, also contains polysaccharides and is strengthened by polymeric lignin covalently cross-linked to hemicellulose.
  • Cellulose is a homopolymer of anhydrocellobiose and thus a linear beta-(1-4)-D-glucan, while hemicelluloses include a variety of compounds, such as xylans, xyloglucans, arabinoxylans, and mannans in complex branched structures with a spectrum of substituents. Although generally polymorphous, cellulose is found in plant tissue primarily as an insoluble crystalline matrix of parallel glucan chains. Hemicelluloses usually hydrogen bond to cellulose, as well as to other hemicelluloses, which help stabilize the cell wall matrix.
  • Cellulose is generally found, for example, in the stems, leaves, hulls, husks, and cobs of plants or leaves, branches, and wood of trees.
  • the cellulosic material can be, but is not limited to, agricultural residue, herbaceous material (including energy crops), municipal solid waste, pulp and paper mill residue, waste paper, and wood (including forestry residue) (see, for example, Wiselogel et al. , 1995, in Handbook on Bioethanol (Charles E. Wyman, editor), pp.
  • the cellulose may be in the form of lignocellulose, a plant cell wall material containing lignin, cellulose, and hemicellulose in a mixed matrix.
  • the cellulosic material is any biomass material.
  • the cellulosic material is lignocellulose, which comprises cellulose, hemicelluloses, and lignin.
  • the cellulosic material is agricultural residue. In another aspect, the cellulosic material is herbaceous material (including energy crops). In another aspect, the cellulosic material is municipal solid waste. In another aspect, the cellulosic material is pulp and paper mill residue. In another aspect, the cellulosic material is waste paper. In another aspect, the cellulosic material is wood (including forestry residue). In another aspect, the cellulosic material is arundo. In another aspect, the cellulosic material is bagasse. In another aspect, the cellulosic material is bamboo. In another aspect, the cellulosic material is corn cob. In another aspect, the cellulosic material is corn fiber.
  • the cellulosic material is corn stover. In another aspect, the cellulosic material is miscanthus. In another aspect, the cellulosic material is orange peel. In another aspect, the cellulosic material is rice straw. In another aspect, the cellulosic material is switchgrass. In another aspect, the cellulosic material is wheat straw.
  • the cellulosic material is aspen. In another aspect, the cellulosic material is eucalyptus. In another aspect, the cellulosic material is fir. In another aspect, the cellulosic material is pine. In another aspect, the cellulosic material is poplar. In another aspect, the cellulosic material is spruce. In another aspect, the cellulosic material is willow.
  • the cellulosic material is algal cellulose. In another aspect, the cellulosic material is bacterial cellulose. In another aspect, the cellulosic material is cotton linter. In another aspect, the cellulosic material is filter paper. In another aspect, the cellulosic material is microcrystalline cellulose. In another aspect, the cellulosic material is phosphoric-acid treated cellulose.
  • the cellulosic material is an aquatic biomass.
  • aquatic biomass means biomass produced in an aquatic environment by a photosynthesis process.
  • the aquatic biomass can be algae, emergent plants, floating-leaf plants, or submerged plants.
  • the cellulosic material may be used as is or may be subjected to pretreatment, using conventional methods known in the art, as described herein. In a preferred aspect, the cellulosic material is pretreated.
  • Acetylxylan esterase means a carboxylesterase (EC 3.1.1.72) that catalyzes the hydrolysis of acetyl groups from polymeric xylan, acetylated xylose, acetylated glucose, alpha-napthyl acetate, and p-nitrophenyl acetate.
  • acetylxylan esterase activity is determined using 0.5 mM p- nitrophenylacetate as substrate in 50 mM sodium acetate pH 5.0 containing 0.01 % TWEENTM 20 (polyoxyethylene sorbitan monolaurate).
  • One unit of acetylxylan esterase is defined as the amount of enzyme capable of releasing 1 ⁇ of p-nitrophenolate anion per minute at pH 5, 25°C.
  • Beta-glucosidase means a beta-D-glucoside glucohydrolase (E.C. 3.2.1 .21 ) that catalyzes the hydrolysis of terminal non-reducing beta-D- glucose residues with the release of beta-D-glucose.
  • beta-glucosidase activity is determined using p-nitrophenyl-beta-D-glucopyranoside as substrate according to the procedure of Venturi et al., 2002, Extracellular beta-D-glucosidase from Chaetomium thermophilum var. coprophilum: production, purification and some biochemical properties, J. Basic Microbiol. 42: 55-66.
  • beta-glucosidase is defined as 1.0 ⁇ of p-nitrophenolate anion produced per minute at 25°C, pH 4.8 from 1 mM p- nitrophenyl-beta-D-glucopyranoside as substrate in 50 mM sodium citrate containing 0.01 % TWEEN® 20 (polyoxyethylene sorbitan monolaurate).
  • Beta-xylosidase means a beta-D-xyloside xylohydrolase (E.C. 3.2.1.37) that catalyzes the exo-hydrolysis of short beta (1 ⁇ 4)- xylooligosaccharides to remove successive D-xylose residues from non-reducing termini.
  • one unit of beta-xylosidase is defined as 1 .0 ⁇ of p-nitrophenolate anion produced per minute at 40°C, pH 5 from 1 mM p-nitrophenyl-beta-D- xyloside as substrate in 100 mM sodium citrate containing 0.01 % TWEEN® 20.
  • Cellobiohydrolase means a 1 ,4-beta-D-glucan cellobiohydrolase (E.C. 3.2.1.91 ) that catalyzes the hydrolysis of 1 ,4-beta-D-glucosidic linkages in cellulose, cellooligosaccharides, or any beta-1 ,4-linked glucose containing polymer, releasing cellobiose from the reducing or non-reducing ends of the chain (Teeri, 1997, Crystalline cellulose degradation: New insight into the function of cellobiohydrolases, Trends in Biotechnology 15: 160-167; Teeri et al., 1998, Trichoderma reesei cellobiohydrolases: why so efficient on crystalline cellulose?, Biochem.
  • E.C. 3.2.1.91 1 ,4-beta-D-glucan cellobiohydrolase
  • Cellulolytic enzyme or cellulase means one or more (e.g., several) enzymes that hydrolyze a cellulosic material. Such enzymes include endoglucanase(s), cellobiohydrolase(s), beta-glucosidase(s), or combinations thereof.
  • the two basic approaches for measuring cellulolytic activity include: (1 ) measuring the total cellulolytic activity, and (2) measuring the individual cellulolytic activities (endoglucanases, cellobiohydrolases, and beta-glucosidases) as reviewed in Zhang et al., Outlook for cellulase improvement: Screening and selection strategies, 2006, Biotechnology Advances 24: 452-481.
  • Total cellulolytic activity is usually measured using insoluble substrates, including Whatman N°1 filter paper, microcrystalline cellulose, bacterial cellulose, algal cellulose, cotton, pretreated lignocellulose, etc.
  • the most common total cellulolytic activity assay is the filter paper assay using Whatman N°1 filter paper as the substrate.
  • cellulolytic enzyme activity is determined by measuring the increase in hydrolysis of a cellulosic material by cellulolytic enzyme(s) under the following conditions: 1 -50 mg of cellulolytic enzyme protein/g of cellulose in PCS (or other pretreated cellulosic material) for 3-7 days at a suitable temperature, e.g., 50°C, 55°C, or 60°C, compared to a control hydrolysis without addition of cellulolytic enzyme protein.
  • Typical conditions are 1 ml reactions, washed or unwashed PCS, 5% insoluble solids, 50 mM sodium acetate pH 5, 1 mM MnS0 4 , 50°C, 55°C, or 60°C, 72 hours, sugar analysis by AMINEX® HPX-87H column (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
  • Endoglucanase means an endo-1 ,4-(1 ,3; 1 ,4)-beta-D- glucan 4-glucanohydrolase (E.C. 3.2.1.4) that catalyzes endohydrolysis of 1 ,4-beta-D- glycosidic linkages in cellulose, cellulose derivatives (such as carboxymethyl cellulose and hydroxyethyl cellulose), lichenin, beta-1 ,4 bonds in mixed beta-1 ,3 glucans such as cereal beta-D-glucans or xyloglucans, and other plant material containing cellulosic components.
  • endoglucanase means an endo-1 ,4-(1 ,3; 1 ,4)-beta-D- glucan 4-glucanohydrolase (E.C. 3.2.1.4) that catalyzes endohydrolysis of 1 ,4-beta-D- glycosidic linkages in cellulose, cellulose derivatives (such
  • Endoglucanase activity can be determined by measuring reduction in substrate viscosity or increase in reducing ends determined by a reducing sugar assay (Zhang et al., 2006, Biotechnology Advances 24: 452-481 ).
  • endoglucanase activity is determined using carboxymethyl cellulose (CMC) as substrate according to the procedure of Ghose, 1987, Pure and Appl. Chem. 59: 257-268, at pH 5, 40°C.
  • Family 61 glycoside hydrolase The term "Family 61 glycoside hydrolase" or
  • “Family GH61” or “GH61” means a polypeptide falling into the glycoside hydrolase Family 61 according to Henrissat B., 1991 , A classification of glycosyl hydrolases based on amino-acid sequence similarities, Biochem. J. 280: 309-316, and Henrissat and Bairoch, 1996, Updating the sequence-based classification of glycosyl hydrolases, Biochem. J. 316: 695-696. The enzymes in this family were originally classified as a glycoside hydrolase family based on measurement of very weak endo-1 ,4-beta-D-glucanase activity in one family member.
  • Hemicellulolytic enzyme or hemicellulase means one or more (e.g., several) enzymes that hydrolyze a hemicellulosic material. See, for example, Shallom and Shoham, 2003, Microbial hemicellulases. Current Opinion In Microbiology 6(3): 219-228). Hemicellulases are key components in the degradation of plant biomass.
  • hemicellulases include, but are not limited to, an acetylmannan esterase, an acetylxylan esterase, an arabinanase, an arabinofuranosidase, a coumaric acid esterase, a feruloyl esterase, a galactosidase, a glucuronidase, a glucuronoyl esterase, a mannanase, a mannosidase, a xylanase, and a xylosidase.
  • the substrates of these enzymes are a heterogeneous group of branched and linear polysaccharides that are bound via hydrogen bonds to the cellulose microfibrils in the plant cell wall, crosslinking them into a robust network. Hemicelluloses are also covalently attached to lignin, forming together with cellulose a highly complex structure. The variable structure and organization of hemicelluloses require the concerted action of many enzymes for its complete degradation.
  • the catalytic modules of hemicellulases are either glycoside hydrolases (GHs) that hydrolyze glycosidic bonds, or carbohydrate esterases (CEs), which hydrolyze ester linkages of acetate or ferulic acid side groups.
  • GHs glycoside hydrolases
  • CEs carbohydrate esterases
  • catalytic modules based on homology of their primary sequence, can be assigned into GH and CE families. Some families, with an overall similar fold, can be further grouped into clans, marked alphabetically (e.g., GH-A).
  • GH-A GH-A
  • a most informative and updated classification of these and other carbohydrate active enzymes is available in the Carbohydrate-Active Enzymes (CAZy) database. Hemicellulolytic enzyme activities can be measured according to Ghose and Bisaria, 1987, Pure & Appl. Chem. 59: 1739-1752, at a suitable temperature, e.g., 50°C, 55°C, or 60°C, and pH, e.g., 5.0 or 5.5.
  • Polypeptide having cellulolytic enhancing activity means a GH61 polypeptide that catalyzes the enhancement of the hydrolysis of a cellulosic material by enzyme having cellulolytic activity.
  • cellulolytic enhancing activity is determined by measuring the increase in reducing sugars or the increase of the total of cellobiose and glucose from the hydrolysis of a cellulosic material by cellulolytic enzyme under the following conditions: 1 -50 mg of total protein/g of cellulose in PCS, wherein total protein is comprised of 50-99.5% w/w cellulolytic enzyme protein and 0.5-50% w/w protein of a GH61 polypeptide having cellulolytic enhancing activity for 1-7 days at a suitable temperature, e.g., 50°C, 55°C, or 60°C, and pH, e.g., 5.0 or 5.5, compared to a control hydrolysis with equal total protein loading without cellulolytic enhancing activity (1 -50 mg of cellulolytic protein/g of cellulose in PCS).
  • suitable temperature e.g., 50°C, 55°C, or 60°C
  • pH e.g., 5.0 or 5.5
  • a mixture of CELLUCLAST® 1.5L (Novozymes A/S, Bagsvaerd, Denmark) in the presence of 2-3% of total protein weight Aspergillus oryzae beta-glucosidase (recombinantly produced in Aspergillus oryzae according to WO 02/095014) or 2-3% of total protein weight Aspergillus fumigatus beta-glucosidase (recombinantly produced in Aspergillus oryzae as described in WO 2002/095014) of cellulase protein loading is used as the source of the cellulolytic activity.
  • the GH61 polypeptides having cellulolytic enhancing activity enhance the hydrolysis of a cellulosic material catalyzed by enzyme having cellulolytic activity by reducing the amount of cellulolytic enzyme required to reach the same degree of hydrolysis preferably at least 1.01-fold, e.g., at least 1 .05-fold, at least 1.10-fold, at least 1.25-fold, at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, or at least 20- fold.
  • Sequence identity The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity”.
  • the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al. , 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later.
  • the parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.
  • the output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:
  • sequence identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 5.0.0 or later.
  • the parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix.
  • the output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:
  • variant means a polypeptide having enzyme activity comprising an alteration, i.e., a substitution, insertion, and/or deletion, at one or more (e.g., several) positions.
  • a substitution means replacement of the amino acid occupying a position with a different amino acid;
  • a deletion means removal of the amino acid occupying a position; and
  • an insertion means adding an amino acid adjacent to and immediately following the amino acid occupying a position.
  • xylanase means a 1 ,4-beta-D-xylan-xylohydrolase (E.C. 3.2.1.8) that catalyzes the endohydrolysis of 1 ,4-beta-D-xylosidic linkages in xylans.
  • xylanase activity is determined with 0.2% AZCL- arabinoxylan as substrate in 0.01 % TRITON® X-100 and 200 mM sodium phosphate buffer pH 6 at 37°C.
  • One unit of xylanase activity is defined as 1.0 ⁇ of azurine produced per minute at 37°C, pH 6 from 0.2% AZCL-arabinoxylan as substrate in 200 mM sodium phosphate pH 6 buffer.
  • the present invention relates to cellulolytic enzyme compositions; the use thereof for hydrolyzing acetylated cellulosic material; and processes of producing fermentation products using a cellulolytic enzyme composition of the invention.
  • the inventors have found that enzyme compositions comprising cellulolytic preparations boost conversion of acetylated cellulosic material. More specifically the inventors surprisingly found that when including acetylxylan esterases (AXEs), in particular acetylxylan esterase derived from Thilavia terrestris, in cellulolytic preparations the cellulose conversion of pretreated acetylated cellulosic material is boosted.
  • AXEs acetylxylan esterases
  • Example 1 shows that acetylxylan esterases (AXEs) boost the glucose and xylose yield when used for cellulolytic hydrolysis of pretreated acetylated corn stover pulp compared to hydrolysis with cellulolytic compositions, but without an acetylxylan esterase (AXE).
  • AXEs acetylxylan esterases
  • the invention relates to an enzyme composition
  • a cellulolytic preparation and an acetylxylan esterase (AXE).
  • AXE acetylxylan esterase
  • the cellulolytic preparation is derived from a strain of Trichoderma, such as a strain of Trichoderma reesei; a strain of Humicola, such as a strain of Humicola insolens, and/or a strain of Chrysosporium, such as a strain of Chrysosporium lucknowense.
  • the cellulolytic preparation is derived from a strain of Trichoderma reesei.
  • the cellulolytic preparation may comprise one or more of the following polypeptides, such as enzymes: GH61 polypeptide having cellulolytic enhancing activity, beta-glucosidase, xylanase, beta-xylosidase, CBHI, CBHII, or a mixture of two, three, four, five or six thereof.
  • the cellulolytic preparation comprises a GH61 polypeptide having cellulolytic enhancing activity and a beta-glucosidase.
  • the cellulolytic preparation comprises a GH61 polypeptide having cellulolytic enhancing activity, a beta-glucosidase, and a xylanase.
  • the cellulolytic preparation comprises a GH61 polypeptide having cellulolytic enhancing activity, a beta-glucosidase, a xylanase and a beta-xylosidase.
  • the cellulolytic preparation comprises a GH61 polypeptide having cellulolytic enhancing activity, a beta-glucosidase, a xylanase, a beta-xylosidase, and a CBHI.
  • the cellulolytic preparation comprises a GH61 polypeptide having cellulolytic enhancing activity, a beta-glucosidase, a xylanase, a beta-xylosidase, a CBHI and a CBHII.
  • enzymes such as endoglucanases, may also be comprises in the cellulolytic preparation.
  • the cellulolytic preparation may in one embodiment comprise one or more beta- glucosidase.
  • the beta-glucosidase may in one embodiment be one derived from a strain of the genus Aspergillus, such as Aspergillus oryzae, such as the one disclosed in WO 2002/095014 or the fusion protein having beta-glucosidase activity disclosed in WO 2008/057637, or Aspergillus fumigates, such as such as one disclosed in WO 2005/047499 or an Aspergillus fumigatus beta-glucosidase variant
  • a beta-glucosidase may also be present or added during hydrolysis.
  • the beta-glucosidase may be an Aspergillus fumigatus beta-glucosidase (e.g., SEQ ID NO: 2 of WO 2005/047499) or a variant thereof disclosed in WO 2012/044915 (hereby incorporated by reference), such as one with the following substitutions: F100D, S283G, N456E, F512Y
  • beta-glucosidase is derived from a strain of the genus Penicillium, such as a strain of the Penicillium brasilianum disclosed in WO 2007/019442, or a strain of the genus Trichoderma, such as a strain of Trichoderma reesei.
  • the cellulolytic preparation may in one embodiment comprise one or more GH61 polypeptide having cellulolytic enhancing activity.
  • the enzyme composition comprises a GH61 polypeptide having cellulolytic enhancing activity, such as one derived from the genus Thermoascus, such as a strain of Thermoascus aurantiacus, such as the one described in WO 2005/074656 as SEQ ID NO: 2; or one derived from the genus Thielavia, such as a strain of Thielavia terrestris, such as the one described in WO 2005/074647 as SEQ ID NO: 7 and SEQ ID NO: 8; or one derived from a strain of Aspergillus, such as a strain of Aspergillus fumigates, such as the one described in WO 2010/138754 as SEQ ID NO: 2; or one derived from a strain derived from Penicillium, such as a strain of Penicillium emersonii
  • the cellulolytic preparation may in one embodiment comprise one or more xylanase.
  • the cellulolytic preparation comprises an xylanase, preferably a GH10 xylanase, such as one derived from a strain of the genus Aspergillus, such as a strain from Aspergillus fumigatus, such as the one disclosed as Xyl III in WO 2006/078256, or Aspergillus aculeatus, such as the one disclosed in WO 94/21785 as SEQ ID NO: 5 (Xyl II).
  • a GH10 xylanase such as one derived from a strain of the genus Aspergillus, such as a strain from Aspergillus fumigatus, such as the one disclosed as Xyl III in WO 2006/078256, or Aspergillus aculeatus, such as the one disclosed in WO 94/21785 as SEQ ID NO: 5 (Xyl II).
  • the cellulolytic preparation may in one embodiment comprise one or more beta- xylosidase.
  • the cellulolytic preparation comprises a beta-xylosidase, such as one derived from a strain of the genus Aspergillus, such as a strain of Aspergillus fumigatus, such as the one disclosed in co-pending US provisional # 61/526,833 or PCT/US12/052163 (Examples 16 and 17), or derived from a strain of Trichoderma, such as a strain of Trichoderma reesei, such as the mature polypeptide of SEQ ID NO: 58 in WO 201 1/057140.
  • a beta-xylosidase such as one derived from a strain of the genus Aspergillus, such as a strain of Aspergillus fumigatus, such as the one disclosed in co-pending US provisional # 61/526,833 or PCT/US12/052163 (Exa
  • the cellulolytic preparation may in one embodiment comprise one or more CBH I (cellobiohydrolase I).
  • the cellulolytic preparation comprises a cellobiohydrolase I (CBHI), such as one derived from a strain of the genus Aspergillus, such as a strain of Aspergillus fumigatus, such as the Cel7A CBHI disclosed in SEQ ID NO: 2 in WO 201 1/057140, or a strain of the genus Trichoderma, such as a strain of Trichoderma reesei.
  • CBHI cellobiohydrolase I
  • the cellulolytic preparation may in one embodiment comprise one or more CBH II (cellobiohydrolase II).
  • the cellobiohydrolase II CBHII
  • CBHII cellobiohydrolase II
  • a strain of the genus Aspergillus such as a strain of Aspergillus fumigatus
  • Trichoderma such as Trichoderma reesei
  • a strain of the genus Thielavia such as a strain of Thielavia terrestris, such as cellobiohydrolase II CEL6A from Thielavia terrestris.
  • the enzyme composition comprises beside the cellulolytic preparation also an acetylxylan esterase (AXE).
  • AXE acetylxylan esterase
  • the acetylxylan esterase (AXE) is derived from a strain of the genus Thielavia, such as a strain of Thielavia terrestris, such as one disclosed in WO 2009/042846 as SEQ ID NO: 2 or SEQ ID NO: 1 herein or an acetylxylan esterase having at least 80%, such as at least 85%, such as at least 90%, preferably 95%, such as at least 96%, such as 97%, such as at least 98%, such as at least 99% identity to SEQ ID NO: 2 in WO 2009/042846 or SEQ ID NO: 1 herein.
  • the acetylxylan esterase is derived from a strain of the genus Aspergillus, such as a strain of Aspergillus aculaetus, such as one disclosed in WO 2010/108918 as SEQ ID NO: 2, or an acetylxylan esterase having at least 80%, such as at least 85%, such as at least 90%, preferably 95%, such as at least 96%, such as 97%, such as at least 98%, such as at least 99% identity to SEQ ID NO: 2 in WO 2010/108918.
  • the acetylxylan esterase is derived from a strain of the genus Aspergillus, such as a strain of Aspergillus aculaetus, such as Aspergillus aculeatus CBS 101.43, such as the one disclosed in WO 95/02689 as SEQ ID NO: 5 or an acetylxylan esterase having at least 80%, such as at least 85%, such as at least 90%, preferably 95%, such as at least 96%, such as 97%, such as at least 98%, such as at least 99% identity to SEQ ID NO: 5 in WO 95/02689.
  • a strain of the genus Aspergillus such as a strain of Aspergillus aculaetus, such as Aspergillus aculeatus CBS 101.43, such as the one disclosed in WO 95/02689 as SEQ ID NO: 5 or an acetylxylan esterase having at least 80%, such as at least 85%,
  • the acetylxylan esterase is derived from a strain of the genus Humicola, such as a strain of Humicola insolens, such as one disclosed in WO 2009/073709 as SEQ ID NO: 2 or as SEQ ID NO: 3 herein or an acetylxylan esterase having at least 80%, such as at least 85%, such as at least 90%, preferably 95%, such as at least 96%, such as 97%, such as at least 98%, such as at least 99% identity to SEQ ID NO: 2 in WO 2009/073709 or as SEQ ID NO: 3 herein.
  • the acetylxylan esterase is derived from a strain of the genus Aspergillus, such as a strain of Aspergillus aculaetus, such as one disclosed in WO 2010/108918 as SEQ ID NO: 2 or as SEQ ID NO: 2 herein or an acetylxylan esterase having at least 80%, such as at least 85%, such as at least 90%, preferably 95%, such as at least 96%, such as 97%, such as at least 98%, such as at least 99% identity to SEQ ID NO: 2 in WO 2010/108918 or as SEQ ID NO: 2 herein.
  • the acetylxylan esterase is derived from is derived from a strain of the genus Thielavia, more preferred a strain of Thielavia terrestris, even more preferred the one disclosed in WO 2009/042846 as SEQ ID NO: 2 or SEQ ID NO: 1 herein.
  • the cellulolytic preparation may comprise a number of difference polypeptides, such as enzymes.
  • the cellulolytic preparation comprises a Trichoderma reesei cellulolytic preparation, further comprising Thermoascus aurantiacus GH61A polypeptide having cellulolytic enhancing activity (WO 2005/074656), Aspergillus oryzae beta- glucosidase fusion protein (WO 2008/057637), and Aspergillus aculeatus xylanase (Xyl II in WO 94/21785).
  • the cellulolytic preparation comprises a Trichoderma reesei cellulolytic preparation, further comprising Thermoascus aurantiacus GH61A polypeptide having cellulolytic enhancing activity (SEQ ID NO: 2 in WO 2005/074656), Aspergillus fumigatus beta-glucosidase (SEQ ID NO: 2 of WO 2005/047499) and Aspergillus aculeatus xylanase (Xyl II disclosed in WO 94/21785).
  • the cellulolytic preparation comprises a Trichoderma reesei cellulolytic preparation, further comprising Thermoascus aurantiacus GH61A polypeptide having cellulolytic enhancing activity (SEQ ID NO: 2 in WO 2005/074656), Aspergillus fumigatus beta-glucosidase (SEQ ID NO: 2 of WO 2005/047499) and Aspergillus aculeatus xylanase (Xyl II disclosed in WO 94/21785) and the acetylxylan esterase (AXE) is the one derived from Thielavia terrestris disclosed in WO 2009/042846 as SEQ I D NO: 2 or SEQ ID NO: 1 herein or an acetylxylan esterase having at least 80%, such as at least 85%, such as at least 90%, preferably at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as
  • the cellulolytic preparation comprises a Trichoderma reesei cellulolytic preparation further comprising Penicillium emersonii GH61A polypeptide having cellulolytic enhancing activity disclosed in WO 201 1/041397, Aspergillus fumigatus beta- glucosidase (SEQ ID NO: 2 of WO 2005/047499) and Aspergillus fumigatus xylanase (Xyl III in WO 2006/078256) and the acetylxylan esterase (AXE) is the one derived from Thielavia terrestris disclosed in WO 2009/042846 as SEQ ID NO: 2 or SEQ ID NO: 1 herein or an acetylxylan esterase having at least 80%, such as at least 85%, such as at least 90%, preferably at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity to SEQ ID NO
  • the enzyme composition of the present invention may be in any form suitable for use, such as, for example, a crude fermentation broth with or without cells removed, a cell lysate with or without cellular debris, a semi-purified or purified enzyme composition, or a host cell, e.g., Trichoderma host cell, as a source of the enzymes.
  • the enzyme composition may be a dry powder or granulate, a non-dusting granulate, a liquid, a stabilized liquid, or a stabilized protected enzyme.
  • Liquid enzyme compositions may, for instance, be stabilized by adding stabilizers such as a sugar, a sugar alcohol or another polyol, and/or lactic acid or another organic acid according to established processes.
  • the acetylxylan esterase and cellulolytic preparation is mixed in a ratio that results in improved hydrolysis of the acetylated lignocellulolytic material.
  • acetylxylan esterase depends on several factors including, but not limited to, the mixture of component cellulolytic and/or hemicellulolytic enzymes, the acetylated cellulosic material, the concentration of acetylated cellulosic material, the pretreatment(s) of the cellulosic material, temperature, time, pH, and inclusion of fermenting organism (e.g., yeast for Simultaneous Saccharification and Fermentation).
  • fermenting organism e.g., yeast for Simultaneous Saccharification and Fermentation
  • an effective amount of cellulolytic preparation added to the acetylated cellulosic material is about 0.01 to about 50.0 mg, e.g., about 1 to about 25 mg, such as about 2 to about 10 mg, such as about 4 to about 8 mg protein per g/DS of the cellulosic material.
  • the acetylxylan esterase (AXE) is used in an amount of, e.g., 0.01 to about 10 mg, such as 0.05 to about 5 mg, such as 0.1 to about 4 mg enzyme protein per DS of the cellulosic material.
  • (AXE) is in the range between 500:1 and 1 :1 , such as between 50:1 and 2:1 , such as around 4: 1.
  • the cellulolytic preparation is derived from Trichoderma reesei and the acetylxylan esterase is derived from a strain of the genus Thielavia, such as a strain of Thielavia terrestris, such as the one disclosed in WO 2009/042846 as SEQ ID NO: 2 or SEQ ID NO: 1 herein or an acetylxylan esterase having at least 80%, such as at least 85%, such as at least 90%, preferably at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity to SEQ ID NO: 2 in WO 2009/042846 or SEQ ID NO: 1 herein, in a ratio of between 500:1 and 1 :1 , such as between 50:1 and 2:1 , such as about 4:1.
  • Cellulosic material is not natively acetylated. However, when cellulosic materials are subjected to pretreatment with, e.g., acids, such as acetic acid, acetylation occurs. Such acetylation impacts enzymatic digestibility of the cellulosic material during hydrolysis.
  • hydrolysis also known as saccharification, the acetylated cellulosic material is hydrolyzed to break down cellulose and/or hemicellulose to fermentable sugars, such as glucose, cellobiose, xylose, xylulose, arabinose, mannose, galactose, and/or soluble oligosaccharides. Hydrolysis may according to the invention be performed enzymatically using an enzyme composition of the present invention.
  • the invention relates to methods of hydrolyzing acetylated cellulosic material comprising subjecting the acetylated cellulosic material to a cellulolytic preparation and an acetylxylan esterase (AXE).
  • AXE acetylxylan esterase
  • the acetylated cellulosic material is pretreated cellulosic material.
  • Acetylated cellulosic material refers to cellulosic material that has a higher degree of acetylation than native cellulosic material.
  • the acetylated cellulosic material may be plant material that comprises cellulosic material (defined above).
  • the acetylated cellulosic material also comprises hemicellulosic material, such as xylans, xyloglucans, arabinoxylans, and mannans in complex branched structures with a spectrum of substituents.
  • the acetylated cellulosic material is plant material chips, plant stem segments and/or whole plant stems.
  • the cellulosic material is selected from the group consisting of arundo, bagasse, bamboo, corn cob, corn fiber, corn stover, miscanthus, orange peel, rice straw, switchgrass, wheat straw.
  • the source of the cellulosic material is corn stover, corn cobs, and/or wheat straw.
  • the acetylated cellulosic material is acetylated corn stover pulp.
  • the cellulosic material can be subjected to particle size reduction, sieving, pre- soaking, wetting, washing, and/or conditioning before pretreatment using methods known in the art.
  • the cellulosic material may have been subjected to conventional pretreatments.
  • Conventional pretreatments include, but are not limited to, steam pretreatment (with or without explosion), acid pretreatment, such as dilute acid pretreatment, hot water pretreatment, alkaline pretreatment, lime pretreatment, wet oxidation, wet explosion, ammonia fiber explosion, organosolv pretreatment, and biological pretreatment.
  • Additional pretreatments include ammonia percolation, ultrasound, electroporation, microwave, supercritical C0 2 , supercritical H 2 0, ozone, ionic liquid, and gamma irradiation pretreatments. Acid pretreatment is preferred.
  • the cellulosic material is preferably pretreated before hydrolysis.
  • the pretreatment can be carried out simultaneously with enzyme hydrolysis to release fermentable sugars, such as glucose, xylose, and/or cellobiose.
  • chemical treatment refers to any chemical pretreatment that promotes the separation and/or release of cellulose, hemicellulose, and/or lignin. Such a pretreatment can convert crystalline cellulose to amorphous cellulose.
  • suitable chemical pretreatment processes include, for example, acid pretreatment, such as dilute acid pretreatment, lime pretreatment, wet oxidation, ammonia fiber/freeze explosion (AFEX), ammonia percolation (APR), ionic liquid, and organosolv pretreatments. Pretreatments including acid pretreatment is preferred.
  • a catalyst such as H 2 S0 4 or S0 2 (typically 0.3 to 5% w/w) is often added prior to steam pretreatment, which decreases the time and temperature, increases the recovery, and improves enzymatic hydrolysis (Ballesteros et al., 2006, Appl. Biochem. Biotechnol. 129- 132: 496-508; Varga et al. , 2004, Appl. Biochem. Biotechnol. 1 13-1 16: 509-523; Sassner et al., 2006, Enzyme Microb. Technol. 39: 756-762).
  • the cellulosic material is mixed with dilute acid, typically H 2 S0 4 , and water to form a slurry, heated by steam to the desired temperature, and after a residence time flashed to atmospheric pressure.
  • dilute acid pretreatment can be performed with a number of reactor designs, e.g., plug-flow reactors, counter-current reactors, or continuous counter-current shrinking bed reactors (Duff and Murray, 1996, supra; Schell et al., 2004, Bioresource Technol. 91 : 179-188; Lee et al., 1999, Adv. Biochem. Eng. Biotechnol. 65: 93-1 15).
  • alkaline pretreatments include, but are not limited to, sodium hydroxide, lime, wet oxidation, ammonia percolation (APR), and ammonia fiber/freeze explosion (AFEX).
  • Lime pretreatment is performed with calcium oxide or calcium hydroxide at temperatures of 85-150°C and residence times from 1 hour to several days (Wyman et al., 2005, Bioresource Technol. 96: 1959-1966; Mosier et al., 2005, Bioresource Technol. 96: 673- 686).
  • WO 2006/110891 , WO 2006/1 10899, WO 2006/1 10900, and WO 2006/1 10901 disclose pretreatment methods using ammonia.
  • Wet oxidation is a thermal pretreatment performed typically at 180-200°C for 5-15 minutes with addition of an oxidative agent such as hydrogen peroxide or over-pressure of oxygen (Schmidt and Thomsen, 1998, Bioresource Technol. 64: 139-151 ; Palonen et al., 2004, Appl. Biochem. Biotechnol. 117: 1-17; Varga et al., 2004, Biotechnol. Bioeng. 88: 567-574; Martin et al., 2006, J. Chem. Technol. Biotechnol. 81 : 1669-1677).
  • the pretreatment is performed preferably at 1-40% dry matter, e.g., 2-30% dry matter or 5-20% dry matter, and often the initial pH is increased by the addition of alkali such as sodium carbonate.
  • Ammonia fiber explosion involves treating the cellulosic material with liquid or gaseous ammonia at moderate temperatures such as 90-150°C and high pressure such as 17- 20 bar for 5-10 minutes, where the dry matter content can be as high as 60% (Gollapalli et al., 2002, Appl. Biochem. Biotechnol. 98: 23-35; Chundawat et al., 2007, Biotechnol. Bioeng. 96: 219-231 ; Alizadeh et al., 2005, Appl. Biochem. Biotechnol. 121 : 1 133-1141 ; Teymouri et al., 2005, Bioresource Technol. 96: 2014-2018).
  • cellulose and hemicelluloses remain relatively intact. Lignin-carbohydrate complexes are cleaved.
  • Organosolv pretreatment delignifies the cellulosic material by extraction using aqueous ethanol (40-60% ethanol) at 160-200°C for 30-60 minutes (Pan et al., 2005, Biotechnol. Bioeng. 90: 473-481 ; Pan et al., 2006, Biotechnol. Bioeng. 94: 851-861 ; Kurabi et al., 2005, Appl. Biochem. Biotechnol. 121 : 219-230). Sulphuric acid is usually added as a catalyst. In organosolv pretreatment, the majority of hemicellulose and lignin is removed.
  • the chemical pretreatment is preferably carried out as acid pretreatment, such as dilute acid treatment, such as a continuous dilute acid treatment.
  • the acid is preferably carried out using acetic acid.
  • other acid such as sulfuric acid, citric acid, nitric acid, phosphoric acid, tartaric acid, succinic acid, hydrogen chloride, or mixtures thereof, can also be used.
  • Mild acid treatment is conducted in the pH range of preferably 1-5, e.g., 1-4 or 1-2.5.
  • the acid concentration is in the range from preferably 0.01 to 10 wt. % acid, e.g., 0.05 to 5 wt. % acid or 0.1 to 2 wt % acid.
  • the acid is contacted with the cellulosic material and held at a temperature, e.g., in the range of preferably 140-200°C, e.g., 165-190°C, for periods ranging from 1 to 60 minutes.
  • pretreatment takes place in an aqueous slurry.
  • the cellulosic material is present during pretreatment in amounts preferably between 10-80 wt. %, e.g., 20-70 wt. % or 30-60 wt. %, such as around 40 wt. %.
  • the pretreated cellulosic material can be unwashed or washed using any method known in the art, e.g., washed with water.
  • mechanical pretreatment or “physical pretreatment” refer to any pretreatment that promotes size reduction of particles.
  • pretreatment can involve various types of grinding or milling (e.g., dry milling, wet milling, or vibratory ball milling).
  • the cellulosic material can be pretreated both physically (mechanically) and chemically.
  • Mechanical or physical pretreatment can, e.g., be coupled with steaming/steam explosion, hydrothermolysis, acid pretreatment, such as dilute or mild acid treatment, high temperature, high pressure treatment, irradiation (e.g., microwave irradiation), or combinations thereof.
  • high pressure means pressure in the range of preferably about 100 to about 400 psi, e.g., about 150 to about 250 psi.
  • high temperature means temperatures in the range of about 100 to about 300°C, e.g., about 140 to about 200°C.
  • mechanical or physical pretreatment is performed in a batch-process using a steam gun hydrolyzer system that uses high pressure and high temperature as defined above, e.g., a Sunds Hydrolyzer available from Sunds Defibrator AB, Sweden.
  • the physical and chemical pretreatments can be carried out sequentially or simultaneously, as desired.
  • biological pretreatment refers to any biological pretreatment that promotes the separation and/or release of cellulose, hemicellulose, and/or lignin from the cellulosic material.
  • Biological pretreatment techniques can involve applying lignin-solubilizing microorganisms and/or enzymes (see, for example, Hsu, T.-A., 1996, Pretreatment of biomass, in Handbook on Bioethanol: Production and Utilization, Wyman, C. E., ed., Taylor & Francis, Washington, DC, 179-212; Ghosh and Singh, 1993, Physicochemical and biological treatments for enzymatic/microbial conversion of cellulosic biomass, Adv. Appl. Microbiol.
  • Preferred pretreatments include chemical pretreatment, a physical pretreatment, or a chemical pretreatment and a physical pretreatment.
  • the cellulosic material is subjected to thermomechanically pulping.
  • the acetylated cellulosic material has been thermomechanically pulped.
  • the acetylated cellulosic material is prepared by pretreatment, includes acid pretreatment.
  • the acetylated cellulosic material has been prepared by pretreating cellulosic material at high temperature, high pressure and/or acid pretreatment.
  • the acid pretreatment is done using acetic acid, or another acid.
  • the acetylated cellulosic material has been prepared by pretreating cellulosic material using organosolv pretreatement, such as Acetosolv and Acetocell processes.
  • the soluble fractions containing sugars, acid(s) and solubilized lignin is removed from the acetylated cellulosic material after pretreatment.
  • hydrolysis of the acetylated cellulosic material is carried out at a temperature between 20-70°C, such as 30-60°C, preferably 45-55°C at a pH in the range 4- 6, such as 4.5-5.5.
  • the acetylated cellulosic material may in an embodiment be present at 1 - 20 (w/w) % of TS, such as 2-10 (w/w) % TS (Total Solids), such as around 5 (w/w) % TS.
  • hydrolysis is carried out for 1-20 days, preferably 5-15 days.
  • hydrolysis is carried out using an enzyme composition of the invention comprising a cellulolytic preparation and an acetylxylan esterase (AXE) as defined in the "Enzyme Composition of the lnvention"-section above.
  • AXE acetylxylan esterase
  • Hydrolysis is carried out in a suitable aqueous environment under conditions that can be readily determined by one skilled in the art.
  • hydrolysis is performed under conditions suitable for the activity of the enzyme(s), i.e., optimal for the enzyme(s).
  • the hydrolysis can be carried out as a fed batch or continuous process where the acetylated cellulosic material is fed gradually to, for example, an enzyme composition containing hydrolysis solution.
  • the hydrolysis i.e., saccharification
  • the temperature is in the range of preferably about 25°C to about 70°C, e.g., about 30°C to about 65°C, about 40°C to about 60°C, or about 50°C to about 55°C.
  • the pH is in the range of preferably about 3 to about 8, e.g., about 3.5 to about 7, about 4 to about 6, or about 5.0 to about 5.5.
  • the dry solids content is in the range of preferably about 5 to about 50 wt. %, e.g., about 10 to about 40 wt. % or about 20 to about 30 wt. %.
  • the invention is directed to processes of using an enzyme composition of the present invention.
  • the invention relates to processes of producing a fermentation product from acetylated cellulosic material, comprising:
  • hydrolysis i.e., saccharification
  • fermentation may be carried out separate or simultaneous.
  • the process of the invention is carried out as separate hydrolysis and fermentation (SHF); simultaneous saccharification and fermentation (SSF); simultaneous saccharification and co-fermentation (SSCF); hybrid hydrolysis and fermentation (HHF); separate hydrolysis and co-fermentation (SHCF); hybrid hydrolysis and co-fermentation (HHCF); or direct microbial conversion (DMC), also sometimes called consolidated bioprocessing (CBP).
  • SHF hydrolysis and fermentation
  • SSF simultaneous saccharification and fermentation
  • SSCF simultaneous saccharification and co-fermentation
  • HHF hybrid hydrolysis and fermentation
  • SHCF separate hydrolysis and co-fermentation
  • HHCF hybrid hydrolysis and co-fermentation
  • DMC direct microbial conversion
  • CBP consolidated bioprocessing
  • SHF uses separate process steps to first enzymatically hydrolyze the acetylated cellulosic material to fermentable sugars, e.g., glucose, cellobiose, and pentose monomers, and then ferment the fermentable sugars to ethanol.
  • fermentable sugars e.g., glucose, cellobiose, and pentose monomers
  • SSCF involves the co-fermentation of multiple sugars (Sheehan, J., and Himmel, M., 1999, Enzymes, energy and the environment: A strategic perspective on the U.S. Department of Energy's research and development activities for bioethanol, Biotechnol. Prog. 15: 817-827).
  • HHF involves a separate hydrolysis step, and in addition a simultaneous saccharification and hydrolysis step, which can be carried out in the same reactor.
  • the steps in an HHF process can be carried out at different temperatures, i.e., high temperature enzymatic hydrolysis (saccharification) followed by SSF at a lower temperature that the fermentation strain can tolerate.
  • DMC combines all three processes (enzyme production, hydrolysis, and fermentation) in one or more (e.g., several) steps where the same organism is used to produce the enzymes for conversion of the cellulosic material to fermentable sugars and to convert the fermentable sugars into a final product.
  • the acetylated cellulosic material may be a material as described in the "Acetylated Cellulosic Materials” section. Hydrolysis is carried in accordance with the "Hydrolysis Methods of the Invention” section. Fermentation
  • the fermentable sugars obtained from the hydrolyzed acetylated cellulosic material can be fermented by one or more (e.g. , several) fermenting microorganisms capable of fermenting the sugars directly or indirectly into a desired fermentation product.
  • Fermentation or “fermentation process” refers to any fermentation process or any process comprising a fermentation step. Fermentation processes also include fermentation processes used in the consumable alcohol industry (e.g. , beer and wine), dairy industry (e.g., fermented dairy products), leather industry, and tobacco industry. The fermentation conditions depend on the desired fermentation product and fermenting organism and can easily be determined by one skilled in the art.
  • sugars released from the cellulosic material as a result of the pretreatment and enzymatic hydrolysis steps, are fermented to a product, e.g., ethanol, by a fermenting organism, such as yeast.
  • Hydrolysis (saccharification) and fermentation can be separate or simultaneous, as described herein.
  • the term 'fermenting microorganism refers to any microorganism, including bacterial and fungal organisms, suitable for use in a desired fermentation process to produce a fermentation product.
  • the fermenting organism can be hexose and/or pentose fermenting organisms, or a combination thereof. Both hexose and pentose fermenting organisms are well known in the art.
  • Suitable fermenting microorganisms are able to ferment, i.e., convert, sugars, such as glucose, xylose, xylulose, arabinose, maltose, mannose, galactose, and/or oligosaccharides, directly or indirectly into the desired fermentation product. Examples of bacterial and fungal fermenting organisms producing ethanol are described by Lin et al., 2006, Appl. Microbiol. Biotechnol. 69: 627-642.
  • yeast examples include bacterial and fungal organisms, such as yeast.
  • Preferred yeast includes strains of Candida, Kluyveromyces, and Saccharomyces, e.g., Candida sonorensis, Kluyveromyces marxianus, and Saccharomyces cerevisiae.
  • Preferred xylose fermenting yeast include strains of Candida, preferably C. sheatae or C. sonorensis; and strains of Pichia, preferably P. stipitis, such as P. stipitis CBS 5773.
  • Preferred pentose fermenting yeast include strains of Pachysolen, preferably P. tannophilus.
  • Organisms not capable of fermenting pentose sugars, such as xylose and arabinose may be genetically modified to do so by methods known in the art.
  • Other fermenting organisms include strains of Bacillus, such as Bacillus coagulans; Candida, such as C. sonorensis, C. methanosorbosa, C. diddensiae, C. parapsilosis, C. naedodendra, C. blankii, C. entomophilia, C. brassicae, C. pseudotropicalis, C. boidinii, C. utilis, and C. scehatae; Clostridium, such as C. acetobutylicum, C. thermocellum, and C. phytofermentans; E. coli, especially E.
  • Geobacillus sp. Hansenula, such as Hansenula anomala
  • Klebsiella such as K. oxytoca
  • Kluyveromyces such as K. marxianus, K. lactis, K. thermotolerans, and K. fragilis
  • Schizosaccharomyces such as S. pombe
  • Thermoanaerobacter such as Thermoanaerobacter saccharolyticum
  • Zymomonas such as Zymomonas mobilis.
  • the yeast is a Bretannomyces. In a more preferred aspect, the yeast is Bretannomyces clausenii. In another preferred aspect, the yeast is a Candida. In another more preferred aspect, the yeast is Candida sonorensis. In another more preferred aspect, the yeast is Candida boidinii. In another more preferred aspect, the yeast is Candida blankii. In another more preferred aspect, the yeast is Candida brassicae. In another more preferred aspect, the yeast is Candida diddensii. In another more preferred aspect, the yeast is Candida entomophiliia. In another more preferred aspect, the yeast is Candida pseudotropicalis. In another more preferred aspect, the yeast is Candida scehatae. In another more preferred aspect, the yeast is Candida utilis.
  • the yeast is a Clavispora. In another more preferred aspect, the yeast is Clavispora lusitaniae. In another more preferred aspect, the yeast is Clavispora opuntiae. In another preferred aspect, the yeast is a Kluyveromyces. In another more preferred aspect, the yeast is Kluyveromyces fragilis. In another more preferred aspect, the yeast is Kluyveromyces marxianus. In another more preferred aspect, the yeast is Kluyveromyces thermotolerans. In another preferred aspect, the yeast is a Pachysolen. In another more preferred aspect, the yeast is Pachysolen tannophilus. In another preferred aspect, the yeast is a Pichia.
  • the yeast is a Pichia stipitis. In another preferred aspect, the yeast is a Saccharomyces spp. In a more preferred aspect, the yeast is Saccharomyces cerevisiae. In another more preferred aspect, the yeast is Saccharomyces distaticus. In another more preferred aspect, the yeast is Saccharomyces uvarum.
  • the bacterium is a Bacillus. In a more preferred aspect, the bacterium is Bacillus coagulans. In another preferred aspect, the bacterium is a Clostridium. In another more preferred aspect, the bacterium is Clostridium acetobutylicum. In another more preferred aspect, the bacterium is Clostridium phytofermentans. In another more preferred aspect, the bacterium is Clostridium thermocellum. In another more preferred aspect, the bacterium is Geobacilus sp. In another more preferred aspect, the bacterium is a Thermoanaerobacter. In another more preferred aspect, the bacterium is Thermoanaerobacter saccharolyticum. In another preferred aspect, the bacterium is a Zymomonas. In another more preferred aspect, the bacterium is Zymomonas mobilis.
  • yeast suitable for ethanol production include, e.g., BIOFERMTM AFT and XR (NABC - North American Bioproducts Corporation, GA, USA), ETHANOL REDTM yeast (Fermentis/Lesaffre, USA), FALITM (Fleischmann's Yeast, USA), FERMIOLTM (DSM Specialties), GERT STRANDTM (Gert Strand AB, Sweden), and SUPERSTARTTM and THERMOSACCTM fresh yeast (Ethanol Technology, Wl, USA).
  • BIOFERMTM AFT and XR NABC - North American Bioproducts Corporation, GA, USA
  • ETHANOL REDTM yeast Fermentis/Lesaffre, USA
  • FALITM Ferischmann's Yeast, USA
  • FERMIOLTM DSM Specialties
  • GERT STRANDTM Gert Strand AB, Sweden
  • SUPERSTARTTM and THERMOSACCTM fresh yeast Ethanol Technology, Wl, USA.
  • the fermenting microorganism has been genetically modified to provide the ability to ferment pentose sugars, such as xylose utilizing, arabinose utilizing, and xylose and arabinose co-utilizing microorganisms.
  • the genetically modified fermenting microorganism is Candida sonorensis. In another preferred aspect, the genetically modified fermenting microorganism is Escherichia coli. In another preferred aspect, the genetically modified fermenting microorganism is Klebsiella oxytoca. In another preferred aspect, the genetically modified fermenting microorganism is Kluyveromyces marxianus. In another preferred aspect, the genetically modified fermenting microorganism is Saccharomyces cerevisiae. In another preferred aspect, the genetically modified fermenting microorganism is Zymomonas mobilis.
  • the fermenting microorganism is typically added to the degraded cellulosic material or hydrolysate and the fermentation is performed for about 8 to about 96 hours, e.g. , about 24 to about 60 hours.
  • the temperature is typically between about 26°C to about 60°C, e.g., about 32°C or 50°C, and about pH 3 to about pH 8, e.g., pH 4-5, 6, or 7.
  • the yeast and/or another microorganism are applied to the degraded cellulosic material and the fermentation is performed for about 12 to about 96 hours, such as typically 24-60 hours.
  • the temperature is preferably between about 20°C to about 60°C, e.g., about 25°C to about 50°C, about 32°C to about 50°C, or about 32°C to about 50°C
  • the pH is generally from about pH 3 to about pH 7, e.g., about pH 4 to about pH 7.
  • some fermenting organisms, e.g., bacteria have higher fermentation temperature optima.
  • Yeast or another microorganism is preferably applied in amounts of approximately 10 5 to 10 12 , preferably from approximately 10 7 to 10 10 , especially approximately 2 x 10 8 viable cell count per ml of fermentation broth. Further guidance in respect of using yeast for fermentation can be found in, e.g., "The Alcohol Textbook” (Editors K. Jacques, T.P. Lyons and D.R. Kelsall, Nottingham University Press, United Kingdom 1999), which is hereby incorporated by reference.
  • the fermented slurry is distilled to extract the ethanol.
  • the ethanol obtained according to the processes of the invention can be used as, e.g., fuel ethanol, drinking ethanol, i.e., potable neutral spirits, or industrial ethanol.
  • a fermentation stimulator can be used in combination with any of the processes described herein to further improve the fermentation process, and in particular, the performance of the fermenting microorganism, such as, rate enhancement and ethanol yield.
  • a "fermentation stimulator” refers to stimulators for growth of the fermenting microorganisms, in particular, yeast.
  • Preferred fermentation stimulators for growth include vitamins and minerals. Examples of vitamins include multivitamins, biotin, pantothenate, nicotinic acid, meso-inositol, thiamine, pyridoxine, para-aminobenzoic acid, folic acid, riboflavin, and Vitamins A, B, C, D, and E.
  • minerals include minerals and mineral salts that can supply nutrients comprising P, K, Mg, S, Ca, Fe, Zn, Mn, and Cu. Fermentation products
  • the term "fermentation product” can be any substance derived from fermentation.
  • the fermentation product can be, without limitation, an alcohol (e.g., arabinitol, n-butanol, isobutanol, ethanol, glycerol, methanol, ethylene glycol, 1 ,3- propanediol [propylene glycol], butanediol, glycerin, sorbitol, and xylitol); an alkane (e.g., pentane, hexane, heptane, octane, nonane, decane, undecane, and dodecane), a cycloalkane (e.g., cyclopentane, cyclohexane, cycloheptane, and cyclooctane), an alkene (e.g.
  • an alcohol e.g., arabinitol, n-butanol, iso
  • pentene, hexene, heptene, and octene an amino acid (e.g., aspartic acid, glutamic acid, glycine, lysine, serine, and threonine); a gas (e.g., methane, hydrogen (H 2 ), carbon dioxide (C0 2 ), and carbon monoxide (CO)); isoprene; a ketone (e.g., acetone); an organic acid (e.g., acetic acid, acetonic acid, adipic acid, ascorbic acid, citric acid, 2,5-diketo-D- gluconic acid, formic acid, fumaric acid, glucaric acid, gluconic acid, glucuronic acid, glutaric acid, 3-hydroxypropionic acid, itaconic acid, lactic acid, malic acid, malonic acid, oxalic acid, oxaloacetic acid, propionic acid, succinic acid, and x
  • the fermentation product is an alcohol.
  • alcohol encompasses a substance that contains one or more (e.g., several) hydroxyl moieties.
  • the alcohol is n-butanol.
  • the alcohol is isobutanol.
  • the alcohol is ethanol.
  • the alcohol is methanol.
  • the alcohol is arabinitol.
  • the alcohol is butanediol.
  • the alcohol is ethylene glycol.
  • the alcohol is glycerin.
  • the alcohol is glycerol.
  • the alcohol is 1 ,3-propanediol. In another more preferred aspect, the alcohol is sorbitol. In another more preferred aspect, the alcohol is xylitol. See, for example, Gong, C. S., Cao, N. J., Du, J., and Tsao, G. T., 1999, Ethanol production from renewable resources, in Advances in Biochemical Engineering/Biotechnology, Scheper, T., ed., Springer-Verlag Berlin Heidelberg, Germany, 65: 207-241 ; Silveira, M. M., and Jonas, R., 2002, The biotechnological production of sorbitol, Appl. Microbiol. Biotechnol.
  • the fermentation product is an alkane.
  • the alkane can be an unbranched or a branched alkane.
  • the alkane is pentane.
  • the alkane is hexane.
  • the alkane is heptane.
  • the alkane is octane.
  • the alkane is nonane.
  • the alkane is decane.
  • the alkane is undecane.
  • the alkane is dodecane.
  • the fermentation product is a cycloalkane.
  • the cycloalkane is cyclopentane.
  • the cycloalkane is cyclohexane.
  • the cycloalkane is cycloheptane.
  • the cycloalkane is cyclooctane.
  • the fermentation product is an alkene.
  • the alkene can be an unbranched or a branched alkene.
  • the alkene is pentene.
  • the alkene is hexene.
  • the alkene is heptene.
  • the alkene is octene.
  • the fermentation product is an amino acid.
  • the organic acid is aspartic acid.
  • the amino acid is glutamic acid.
  • the amino acid is glycine.
  • the amino acid is lysine.
  • the amino acid is serine.
  • the amino acid is threonine. See, for example, Richard and Margaritis, 2004, Empirical modeling of batch fermentation kinetics for poly(glutamic acid) production and other microbial biopolymers, Biotechnology and Bioengineering 87(4): 501-515.
  • the fermentation product is a gas.
  • the gas is methane.
  • the gas is H 2 .
  • the gas is C0 2 .
  • the gas is CO. See, for example, Kataoka, Miya, and Kiriyama, 1997, Studies on hydrogen production by continuous culture system of hydrogen-producing anaerobic bacteria, Water Science and Technology 36(6-7): 41-47; and Gunaseelan, 1997, Anaerobic digestion of biomass for methane production: A review, Biomass and Bioenergy, 13(1-2): 83-1 14.
  • the fermentation product is isoprene.
  • the fermentation product is a ketone.
  • ketone encompasses a substance that contains one or more (e.g., several) ketone moieties.
  • the ketone is acetone. See, for example, Qureshi and Blaschek, 2003, supra.
  • the fermentation product is an organic acid.
  • the organic acid is acetic acid.
  • the organic acid is acetonic acid.
  • the organic acid is adipic acid.
  • the organic acid is ascorbic acid.
  • the organic acid is citric acid.
  • the organic acid is 2,5-diketo-D-gluconic acid.
  • the organic acid is formic acid.
  • the organic acid is fumaric acid.
  • the organic acid is glucaric acid.
  • the organic acid is gluconic acid.
  • the organic acid is glucuronic acid.
  • the organic acid is glutaric acid. In another preferred aspect, the organic acid is 3-hydroxypropionic acid. In another more preferred aspect, the organic acid is itaconic acid. In another more preferred aspect, the organic acid is lactic acid. In another more preferred aspect, the organic acid is malic acid. In another more preferred aspect, the organic acid is malonic acid. In another more preferred aspect, the organic acid is oxalic acid. In another more preferred aspect, the organic acid is propionic acid. In another more preferred aspect, the organic acid is succinic acid. In another more preferred aspect, the organic acid is xylonic acid. See, for example, Chen and Lee, 1997, Membrane-mediated extractive fermentation for lactic acid production from cellulosic biomass, Appl. Biochem. Biotechnol. 63-65: 435-448. Recovery
  • the fermentation product(s) are optionally recovered after fermentation using any method known in the art including, but not limited to, chromatography, electrophoretic procedures, differential solubility, distillation, or extraction.
  • alcohol such as ethanol
  • Ethanol with a purity of up to about 96 vol. % can be obtained, which can be used as, for example, fuel ethanol, drinking ethanol, i.e., potable neutral spirits, or industrial ethanol.
  • Paragraph 1 An enzyme composition comprising a cellulolytic preparation and an acetylxylan esterase (AXE).
  • Paragraph 2 The enzyme composition of paragraph 1 , wherein the cellulolytic preparation is derived from Trichoderma reesei, Humicola insolens or Chrysosporium lucknowense.
  • Paragraph 3 The enzyme composition of paragraph 1 or 2, wherein the cellulolytic preparation comprises a beta-glucosidase, preferably one derived from a strain of the genus Aspergillus, such as Aspergillus oryzae, such as the one disclosed in WO 2002/095014 or the fusion protein having beta-glucosidase activity disclosed in WO 2008/057637, or Aspergillus fumigates, such as such as one disclosed in WO 2005/047499 or an Aspergillus fumigatus beta-glucosidase variant disclosed in co-pending US provisional application no.
  • a beta-glucosidase preferably one derived from a strain of the genus Aspergillus, such as Aspergillus oryzae, such as the one disclosed in WO 2002/095014 or the fusion protein having beta-glucosidase activity disclosed in WO 2008/057637, or Aspergillus fumigates, such as such as one
  • 61/388,997 or WO 2012/044915 with the following substitutions: F100D, S283G, N456E, F512Y; or a strain of the genus a strain Penicillium, such as a strain of the Penicillium brasilianum disclosed in WO 2007/019442, or a strain of the genus Trichoderma, such as a strain of Trichoderma reesei. Paragraph 4.
  • the cellulolytic preparation comprises a GH61 polypeptide having cellulolytic enhancing activity such as one derived from the genus Thermoascus, such as a strain of Thermoascus aurantiacus, such as the one described in WO 2005/074656 as SEQ ID NO: 2; or one derived from the genus Thielavia, such as a strain of Thielavia terrestris, such as the one described in WO 2005/074647 as SEQ ID NO: 7 and SEQ ID NO: 8; or one derived from a strain of Aspergillus, such as a strain of Aspergillus fumigates, such as the one described in WO 2010/138754 as SEQ ID NO: 1 and SEQ ID NO: 2; or one derived from a strain derived from Penicillium, such as a strain of Penicillium emersonii, such as the one disclosed in WO
  • Paragraph 5 The enzyme composition of any of paragraphs 1 -4, wherein the cellulolytic preparation comprises a xylanase, preferably a GH10 xylanase, such as one derived from a strain of the genus Aspergillus, such as a strain from Aspergillus fumigatus, such as the one disclosed as Xyl III in WO 2006/078256, or Aspergillus aculeatus, such as the one disclosed in WO 94/21785 as SEQ ID NO: 5 (Xyl II).
  • a xylanase preferably a GH10 xylanase, such as one derived from a strain of the genus Aspergillus, such as a strain from Aspergillus fumigatus, such as the one disclosed as Xyl III in WO 2006/078256, or Aspergillus aculeatus, such as the one disclosed in WO 94/21785 as SEQ ID NO:
  • the cellulolytic preparation comprises a beta-xylosidase, such as one derived from a strain of the genus Aspergillus, such as a strain of Aspergillus fumigatus, such as the one disclosed in copending US provisional # 61/526833 or PCT/US 12/052163 (Examples 16 and 17), or derived from a strain of Trichoderma, such as a strain of Trichoderma reesei, such as the mature polypeptide of SEQ ID NO: 58 in WO 201 1/057140.
  • a beta-xylosidase such as one derived from a strain of the genus Aspergillus, such as a strain of Aspergillus fumigatus, such as the one disclosed in copending US provisional # 61/526833 or PCT/US 12/052163 (Examples 16 and 17)
  • a strain of Trichoderma such as a strain of Trichoderma reesei
  • Paragraph 7 The enzyme composition of any of paragraphs 1 -6, wherein the cellulolytic preparation comprises a cellobiohydrolase I (CBH I), such as one derived from a strain of the genus Aspergillus, such as a strain of Aspergillus fumigatus, such as the Cel7a CBHI disclosed in SEQ ID NO: 2 in WO 201 1/057140, or a strain of the genus Trichoderma, such as a strain of Trichoderma reesei.
  • CBH I cellobiohydrolase I
  • Paragraph 8 The enzyme composition of any of paragraphs 1 -7, wherein the cellulolytic preparation comprises a cellobiohydrolase II (CBH II, such as one derived from a strain of the genus Aspergillus, such as a strain of Aspergillus fumigates; or a strain of the genus Trichoderma, such as Trichoderma reesei, or a strain of the genus Thielavia, such as a strain of Thielavia terrestris, such as cellobiohydrolase II CEL6A from Thielavia terrestris.
  • CBH II cellobiohydrolase II
  • acetylxylan esterase is derived from a strain of the genus Thielavia, such as a strain of Thielavia terrestris, such as one disclosed in WO 2009/042846 as SEQ ID NO: 2 or SEQ ID NO: 1 herein or an acetylxylan esterase having at least 80%, such as at least 85%, such as at least 90%, preferably 95%, such as at least 96%, such as 97%, such as at least 98%, such as at least 99% identity to SEQ ID NO: 2 in WO 2009/042846 or SEQ ID NO: 1 herein.
  • AXE acetylxylan esterase
  • Paragraph 10 The enzyme composition of any of paragraphs 1 -8, wherein the acetylxylan esterase (AXE) is derived from a strain of the genus Aspergillus, such as a strain of Aspergillus aculaetus, such as one disclosed in WO 2010/108918 as SEQ ID NO: 2 or an acetylxylan esterase having at least 80%, such as at least 85%, such as at least 90%, preferably 95%, such as at least 96%, such as 97%, such as at least 98%, such as at least 99% identity to SEQ ID NO: 2 in WO 2010/108918.
  • AXE acetylxylan esterase
  • Paragraph 1 The enzyme composition of any of paragraphs 1 -8, wherein the acetylxylan esterase (AXE) is derived from a strain of the genus Aspergillus, such as a strain of Aspergillus aculaetus, such as Aspergillus aculeatus CBS 101.43, such as the one disclosed in WO 1995/002689 as SEQ ID NO: 5 or an acetylxylan esterase having at least 80%, such as at least 85%, such as at least 90%, preferably 95%, such as at least 96%, such as 97%, such as at least 98%, such as at least 99% identity to SEQ ID NO: 5 in WO 1995/002689.
  • AXE acetylxylan esterase
  • acetylxylan esterase is derived from a strain of the genus Humicola such as a strain of Humicola insolens, such as one disclosed in WO 2009/073709 as SEQ ID NO: 2 or as SEQ ID NO: 3 herein or an acetylxylan esterase having at least 80%, such as at least 85%, such as at least 90%, preferably 95%, such as at least 96%, such as 97%, such as at least 98%, such as at least 99% identity to SEQ ID NO: 2 in WO 2009/073709 or as SEQ ID NO: 3 herein.
  • AXE acetylxylan esterase
  • Paragraph 13 The enzyme composition of any of paragraphs 1 -12, wherein the cellulolytic preparation comprises a GH61 polypeptide having cellulolytic enhancing activity and a beta- glucosidase.
  • Paragraph 14 The enzyme composition of any of paragraphs 1 -12, wherein the cellulolytic preparation comprises a GH61 polypeptide having cellulolytic enhancing activity, a beta- glucosidase, and a xylanase.
  • Paragraph 15 The enzyme composition of any of paragraphs 1 -12, wherein the cellulolytic preparation comprises a GH61 polypeptide having cellulolytic enhancing activity, a beta- glucosidase, a xylanase and a beta-xylosidase.
  • Paragraph 16 The enzyme composition of any of paragraphs 1 -12, wherein the cellulolytic preparation comprises a GH61 polypeptide having cellulolytic enhancing activity, a beta- glucosidase, a xylanase, a beta-xylosidase, and a CBHI.
  • Paragraph 17 The enzyme composition of any of paragraphs 1 -12, wherein the cellulolytic preparation comprises a GH61 polypeptide having cellulolytic enhancing activity, a beta- glucosidase, a xylanase, a beta-xylosidase, a CBHI and a CBHII.
  • Paragraph 18 The enzyme composition of any of paragraphs 1 -17, wherein the cellulolytic preparation is a Trichoderma reesei cellulolytic preparation, further comprising Thermoascus aurantiacus GH61A polypeptide having cellulolytic enhancing activity (SEQ ID NO: 2 in WO 2005/074656), Aspergillus oryzae beta-glucosidase fusion protein (WO 2008/057637), and Aspergillus aculeatus xylanase (Xyl II in WO 94/21785).
  • SEQ ID NO: 2 in WO 2005/074656
  • Aspergillus oryzae beta-glucosidase fusion protein WO 2008/057637
  • Aspergillus aculeatus xylanase Xyl II in WO 94/21785).
  • Thermoascus aurantiacus GH61A polypeptide having cellulolytic enhancing activity SEQ ID NO: 2 in WO 2005/074656
  • Aspergillus fumigatus beta-glucosidase SEQ ID NO: 2 of WO 2005/047499
  • Aspergillus aculeatus xylanase Xyl II disclosed in WO
  • Paragraph 20 The enzyme composition of any of paragraphs 1 -17, wherein the cellulolytic preparation is a Trichoderma reesei cellulolytic preparation further comprising Thermoascus aurantiacus GH61A polypeptide having cellulolytic enhancing activity (SEQ ID NO: 2 in WO 2005/074656), Aspergillus fumigatus beta-glucosidase (SEQ ID NO: 2 of WO 2005/047499) and Aspergillus aculeatus xylanase (Xyl II disclosed in WO 94/21785) and the acetylxylan esterase (AXE) is the one derived from Thielavia terrestris disclosed in WO 2009/042846 as SEQ ID NO: 2 or SEQ ID NO: 1 herein or an acetylxylan esterase having at least 80%, such as at least 85%, such as at least 90%, preferably at least 95%, such as at least 96%, such as at
  • Paragraph 21 The enzyme composition of any of paragraphs 1 -17, wherein the cellulolytic preparation is a Trichoderma reesei cellulolytic preparation further comprising Penicillium emersonii GH61A polypeptide having cellulolytic enhancing activity disclosed in WO 201 1/041397, Aspergillus fumigatus beta-glucosidase (SEQ ID NO: 2 of WO 2005/047499) and Aspergillus fumigatus xylanase (Xyl III in WO 2006/078256) and the acetylxylan esterase (AXE) is the one derived from Thielavia terrestris disclosed in WO 2009/042846 as SEQ ID NO: 2 or SEQ ID NO: 1 or an acetylxylan esterase having at least 80%, such as at least 85%, such as at least 90%, preferably at least 95%, such as at least 96%, such as at least 97%, such as at least 98%
  • Paragraph 22 The enzyme composition of any of paragraphs 1 -21 , wherein the ratio between cellulolytic preparation and acetylxylan esterase (AXE) is in the range between 500:1 and 100:1 , such as between 50:1 and 2:1 , such as around 4:1.
  • AXE acetylxylan esterase
  • Paragraph 23 A method of hydrolyzing acetylated cellulosic material, comprising subjecting the acetylated cellulosic material to a cellulolytic preparation and an acetylxylan esterase (AXE).
  • Paragraph 24 The method of paragraph 23, wherein acetylated cellulosic material is pretreated cellulosic material.
  • Paragraph 25 The method of paragraph 23 or 24, wherein the cellulosic material is plant material chips, plant stem segments and/or whole plant stems.
  • Paragraph 26 The method of paragraph 25, wherein cellulosic material is selected from the group comprising arundo, bagasse, bamboo, corn cob, corn fiber, corn stover, miscanthus, orange peel, rice straw, switchgrass, wheat straw.
  • Paragraph 27 The method of paragraph 26, wherein the source of the cellulosic material is corn stover, corn cobs, and/or wheat straw.
  • Paragraph 28 The method of any of paragraphs 24-27, wherein the pretreating cellulosic material is pretreated by chemical pretreatment, a physical pretreatment, or a chemical pretreatment and a physical pretreatment.
  • Paragraph 29 The method of any of paragraphs 24-28, wherein the cellulosic material is thermomechamically pulped plant material.
  • Paragraph 30 The method of any of paragraphs 24-29, wherein the acetylated cellulosic material is thermomechanically pulped plant material, such as acetylated corn stover pulp.
  • Paragraph 31 The method of any of paragraphs 24-30, wherein pretreating the cellulosic material includes pretreatment with an acid.
  • Paragraph 32 The method of any of paragraphs 24-31 , wherein the acetylated cellulosic material has been prepared by pretreating cellulosic material at high temperature, high pressure with an acid.
  • Paragraph 33 The method of paragraph 31 or 32, wherein acid pretreatment is carried out using acetic acid.
  • Paragraph 34 The method of any of paragraphs 24-33, wherein the acetylated cellulosic material has been prepared by pretreating cellulosic material using organosolv pretreatement, such as Acetosolv and Acetocell processes.
  • Paragraph 35 The method of any of paragraphs 24-34, wherein the soluble fractions containing sugars, acid and solubilized lignin is removed from the acetylated cellulosic material after pretreatment.
  • Paragraph 36 The method of any of paragraphs 24-35, wherein hydrolysis is carried out at a temperature between 20-70°C, such as 30-60°C, preferably 45-55°C at a pH in the range 4- 6, such as 4.5-5.5.
  • Paragraph 37 The method of any of paragraph 24-36, wherein the cellulosic material is present at 1-20 (w/w) % of TS, such as 2-10 (w/w)% TS, such as around 5 (w/w) % TS.
  • Paragraph 38 The method of any of paragraphs 24-37, wherein the hydrolysis is carried out for 1 -20 days, preferably between from 5-15 days.
  • Paragraph 39 The method of any of paragraphs 24-38, wherein the cellulolytic preparation is derived from Trichoderma reesei, Humicola insolens or Chrysosporium lucknowense.
  • Paragraph 40 The method of any of paragraphs 24-39, wherein the cellulolytic preparation comprises a beta-glucosidase, preferably one derived from a strain of the genus Aspergillus, such as Aspergillus oryzae, such as the one disclosed in WO 2002/095014 or the fusion protein having beta-glucosidase activity disclosed in WO 2008/057637, or Aspergillus fumigatus, such as such as one disclosed in WO 2005/047499 or an Aspergillus fumigatus beta-glucosidase variant disclosed in co-pending US provisional application # 61/388,997 or WO 2012/044915 with the following substitutions: F100D, S283G, N456E, F512Y; or a strain of the genus a strain Penicillium, such as a strain of the Penicillium brasilianum disclosed in WO 2007/019442, or a strain of the genus Trichoderma, such as a
  • the cellulolytic preparation comprises a GH61 polypeptide having cellulolytic enhancing activity, such as one derived from the genus Thermoascus, such as a strain of Thermoascus aurantiacus, such as the one described in WO 2005/074656 as SEQ ID NO: 2; or one derived from the genus Thielavia, such as a strain of Thielavia terrestris, such as the one described in WO 2005/074647 as SEQ ID NO: 7 and SEQ ID NO: 8; or one derived from a strain of Aspergillus, such as a strain of Aspergillus fumigates, such as the one as described in WO 2010/138754 as SEQ ID NO: 1 and SEQ ID NO: 2; or one derived from a strain derived from Penicillium, such as a strain of Penicillium emersonii, such as the
  • Paragraph 42 The method of any of paragraphs 24-41 , wherein the cellulolytic preparation comprises a xylanase, preferably a GH10 xylanase, such as one derived from a strain of the genus Aspergillus, such as a strain from Aspergillus fumigatus, such as the one disclosed as Xyl III in WO 2006/078256, or Aspergillus aculeatus, such as the one disclosed in WO 94/21785 as SEQ ID NO: 5 (Xyl II).
  • a xylanase preferably a GH10 xylanase, such as one derived from a strain of the genus Aspergillus, such as a strain from Aspergillus fumigatus, such as the one disclosed as Xyl III in WO 2006/078256, or Aspergillus aculeatus, such as the one disclosed in WO 94/21785 as SEQ ID NO: 5
  • the cellulolytic preparation comprises a beta-xylosidase, such as one derived from a strain of the genus Aspergillus, such as a strain of Aspergillus fumigatus, such as the one disclosed in co-pending US provisional # 61/526833 or PCT/US12/052163, or derived from a strain of Trichoderma, such as a strain of Trichoderma reesei, such as the mature polypeptide of SEQ 10 NO: 58 in WO 201 1/057140.
  • a beta-xylosidase such as one derived from a strain of the genus Aspergillus, such as a strain of Aspergillus fumigatus, such as the one disclosed in co-pending US provisional # 61/526833 or PCT/US12/052163, or derived from a strain of Trichoderma, such as a strain of Trichoderma reesei, such as the mature polypeptide of SEQ 10 NO
  • Paragraph 44 The method of any of paragraphs 24-42, wherein the acetylxylan esterase (AXE) is derived from a strain of the genus Thielavia, such as a strain of Thielavia terrestris, such as one disclosed in WO 2009/042846 as SEQ ID NO: 2 or SEQ ID NO: 1 herein or an acetylxylan esterase having at least 80%, such as at least 85%, such as at least 90%, preferably at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity to SEQ I D NO: 2 in WO 2009/042846.
  • AXE acetylxylan esterase
  • Paragraph 45 The method of any of paragraphs 24-43, wherein the acetylxylan esterase (AXE) is derived from a strain of the genus Aspergillus, such as a strain of Aspergillus aculaetus, such as one disclosed in WO 2010/108918 as SEQ ID NO: 2 or SE ID NO: 2 herein or an acetylxylan esterase having at least 80%, such as at least 85%, such as at least 90%, preferably 95%, such as at least 96%, such as 97%, such as at least 98%, such as at least 99% identity to SEQ ID NO: 2 in WO 2010/108918 or SEQ ID NO: 2 herein.
  • AXE acetylxylan esterase
  • Paragraph 46 The method of any of paragraphs 24-43, wherein the acetylxylan esterase (AXE) is derived from a strain of the genus Aspergillus, such as a strain of Aspergillus aculaetus, such as Aspergillus aculeatus CBS 101 .43, such as the one disclosed in WO 1995/002689 as SEQ ID NO: 5 or an acetylxylan esterase having at least 80%, such as at least 85%, such as at least 90%, preferably 95%, such as at least 96%, such as 97%, such as at least 98%, such as at least 99% identity to SEQ ID NO: 5 in WO 1995/002689.
  • Paragraph 47 Paragraph 47.
  • acetylxylan esterase is derived from a strain of the genus Humicola such as a strain of Humicola insolens, such as one disclosed in WO 2009/073709 as SEQ ID NO: 2 or as SEQ ID NO: 3 herein or an acetylxylan esterase having at least 80%, such as at least 85%, such as at least 90%, preferably 95%, such as at least 96%, such as 97%, such as at least 98%, such as at least 99% identity to SEQ ID NO: 2 in WO 2009/073709 or as SEQ I D NO: 3 herein.
  • Paragraph 48 The method of any of paragraphs 24-47, wherein the cellulolytic preparation comprises a cellobiohydrolase I (CBHI), such as one derived from a strain of the genus Aspergillus, such as a strain of Aspergillus fumigatus, or a strain of the genus Trichoderma, such as a strain of Trichoderma reesei.
  • CBHI cellobiohydrolase I
  • Paragraph 49 The method of any of paragraphs 24-48, wherein the cellulolytic preparation comprises a cellobiohydrolase II (CBHII), such as one derived from Aspergillus fumigates; or a strain of the genus Trichoderma, such as Trichoderma reesei, or a strain of the genus Thielavia, such as a strain of Thielavia terrestris, such as cellobiohydrolase II CEL6A from Thielavia terrestris.
  • CBHII cellobiohydrolase II
  • Paragraph 50 The method of any of paragraphs 24-49, wherein the cellulolytic preparation comprises a GH61 polypeptide having cellulolytic enhancing activity and a beta-glucanase.
  • Paragraph 51 The method of any of paragraphs 24-49, wherein the cellulolytic preparation comprises a GH61 polypeptide having cellulolytic enhancing activity, a beta-glucanase, and a xylanase.
  • Paragraph 52 The method of any of paragraphs 24-49, wherein the cellulolytic preparation comprises a GH61 polypeptide having cellulolytic enhancing activity, a beta-glucanase, a xylanase and a beta-xylosidase.
  • Paragraph 53 The method of any of paragraphs 24-49, wherein the cellulolytic preparation comprises a GH61 polypeptide having cellulolytic enhancing activity, a beta-glucanase, a xylanase, a beta-xylosidase, and a CBHI.
  • Paragraph 54 The method of any of paragraphs 24-49, wherein the cellulolytic preparation comprises a GH61 polypeptide having cellulolytic enhancing activity, a beta-glucanase, a xylanase, a beta-xylosidase, a CBHI and a CBHII.
  • Paragraph 55 The method of any of paragraphs 24-49, wherein the cellulolytic preparation comprises a GH61 polypeptide having cellulolytic enhancing activity, a beta-glucanase, a xylanase, a beta-xylosidase, a CBHI and a CBHII.
  • the cellulolytic preparation is a Trichoderma reesei cellulolytic preparation, further comprising Thermoascus aurantiacus GH61A polypeptide having cellulolytic enhancing activity (SEQ ID NO: 1 and SEQ ID NO: 2 in WO 2005/074656), Aspergillus oryzae beta-glucosidase fusion protein (WO 2008/057637), and Aspergillus aculeatus xylanase (Xyl II in WO 94/21785).
  • Paragraph 56 The method of any of paragraphs 24-55, wherein the cellulolytic preparation is a Trichoderma reesei cellulolytic preparation, further comprising Thermoascus aurantiacus GH61A polypeptide having cellulolytic enhancing activity (SEQ ID NO: 2 in WO 2005/074656), Aspergillus fumigatus beta-glucosidase (SEQ ID NO: 2 of WO 2005/047499) and Aspergillus aculeatus xylanase (Xyl I I disclosed in WO 94/21785).
  • Thermoascus aurantiacus GH61A polypeptide having cellulolytic enhancing activity SEQ ID NO: 2 in WO 2005/074656
  • Aspergillus fumigatus beta-glucosidase SEQ ID NO: 2 of WO 2005/047499
  • Aspergillus aculeatus xylanase Xyl I I disclosed
  • Paragraph 57 The method of any of paragraphs 24-56, wherein the cellulolytic preparation is a Trichoderma reesei cellulolytic preparation, further comprising Penicillium emersonii GH61A polypeptide having cellulolytic enhancing activity (SEQ ID NO: 2 in WO 201 1/04139, Aspergillus fumigatus beta-glucosidase variant (disclosed in US provisional application No.
  • Paragraph 58 The method of any of paragraphs 24-57, wherein the cellulolytic preparation is added in amounts of about 0.01 to about 50.0 mg, e.g., about 1 to about 25 mg, such as about 2-10 mg, such as about 4 to about 8 mg protein per g/DS of the cellulosic material.
  • Paragraph 59 The method of any of paragraphs 24-58, wherein the acetylxylan esterase (AXE) is used in amounts of 0.01 to about 10 mg, such as 0.05 to about 5 mg, such as 0.1 to about 4 mg enzyme protein per g/DS of the cellulosic material.
  • AXE acetylxylan esterase
  • Paragraph 60 The method of any of paragraphs 24-59, wherein the ratio between cellulolytic preparation and acetylxylan esterase (AXE) is in the range in a ratio of between 500:1 and 1 :1 , such as between 50:1 and 2:1 , such as about 4:1.
  • AXE acetylxylan esterase
  • Paragraph 61 The method of any of paragraphs 24-60, wherein the cellulolytic preparation is a Trichoderma reesei cellulolytic preparation further comprising Thermoascus aurantiacus GH61A polypeptide having cellulolytic enhancing activity (SEQ ID NO: 1 and SEQ ID NO: 2 in WO 2005/074656), Aspergillus fumigatus beta-glucosidase (SEQ ID NO: 2 of WO 2005/047499) and Aspergillus aculeatus xylanase (Xyl II disclosed in WO 94/21785), further comprising and the acetylxylan esterase (AXE) derived from Thielavia terrestris disclosed in WO 2009/042846 as SEQ ID NO: 2 or SEQ ID NO: 1 herein or an acetylxylan esterase having at least 80%, such as at least 85%, such as at least 90%, preferably at least 95%, such as
  • Paragraph 62 The method of any of paragraphs 24-61 , wherein the cellulolytic preparation is a Trichoderma reesei cellulolytic preparation further comprising Penicillium emersonii GH61A polypeptide having cellulolytic enhancing activity disclosed in WO 201 1/041397, Aspergillus fumigatus beta-glucosidase variant (disclosed in co-pending US provisional application # 61/388,997 or WO 2012/044915 with the following substitutions: F100D, S283G, N456E, F512Y, Aspergillus fumigatus xylanase (Xyl III in WO 2006/078256), further comprising the acetylxylan esterase (AXE) derived from Thielavia terrestris disclosed in WO 2009/042846 as SEQ ID NO: 2 or SEQ ID NO: 1 herein or an acetylxylan esterase having at least 80%, such as at least
  • Paragraph 63 A process of producing a fermentation product from acetylated cellulosic material, comprising:
  • Paragraph 64 The process of paragraph 63, wherein the fermentation product is ethanol.
  • Paragraph 65 The process of paragraph 63 or 64, wherein the fermenting organism is a yeast, such as strain of the genus Saccharomyces, such as a strain of Saccharomyce cerevisie.
  • Paragraph 66 The process of any of paragraphs 63-65, wherein the cellulosic material is corn stover pulp. Materials & Methods
  • Acetylated corn stover pulp containing about 75% cellulose and 1 1 % xylan was obtained from Archer Daniels Midland (ADM), USA.
  • Carbohydrates and acetate groups can be measured as per NREL methods (see A.
  • Acetylated corn stover pulp containing about 75% cellulose and 1 1 % xylan was used. The degree of acetylation based on overall carbohydrates was measured to be around 8% (1 acetyl group per 12.5 sugar units).
  • Corn stover pulp was prepared by pretreating corn stover at high temperature and pressure using acetic acid. The resulting slurry was put through a solid/liquid separator (pressure filtration) to remove soluble fractions containing sugars, acetic acid and solubilized lignin.
  • the insoluble substrate (acetylated pulp) was washed with tap water to a pH above 5.0 and was subjected to enzymatic hydrolysis in a 24 well (5 mL) polypropylene cell growth plates (Whatman Uniplate) in quadruplicates at a 5 g hydrolysis scale, 2.5% solids, 50 mM citrate buffer, pH of 5. l and 50°C for 1 1 days using i) Trichoderma reesei cellulolytic preparation, Thermoascus aurantiacus GH61A polypeptide having cellulolytic enhancing activity (described in WO 2005/074656 as SEQ ID
  • the sugar concentrations of samples diluted in 0.005 M H 2 S0 4 were measured using a 4.6 x 250 mm AMINEX® HPX-87H column (Bio-Rad Laboratories, Inc., Hercules, CA, USA) by elution with 0.005 M H 2 S0 4 at 65°C at a flow rate of 0.6 ml per minute, and quantitation by integration of the glucose, cellobiose, and xylose signals from refractive index detection (CHEMSTATION®, AGILENT® 1 100 HPLC, Agilent Technologies, Santa Clara, CA, USA) calibrated by pure sugar samples. Glucose and xylose yields were calculated as % of theoretical based on initial cellulose and xylan input and concentrations of glucose and xylose obtained after enzyme hydrolysis.
  • the acetylated corn stover pulp containing about 75% cellulose and 1 1 % xylan was used.
  • the degree of acetylation based on overall carbohydrates was measured to be around 8% (1 acetyl group per 12.5 sugar units).
  • Corn stover pulp was prepared by pretreating corn stover at high temperature and pressure using acetic acid. The resulting slurry was put through a S/L separator (pressure filtration) to remove soluble fractions containing sugars, acetic acid and solubilized lignin.
  • the insoluble substrate (acetylated pulp) was washed with tap water to a pH above 5.0 and was subjected to enzymatic hydrolysis in a rotisserie incubator in duplicates at a 20 g hydrolysis scale, 5% solids, 50 mM citrate buffer, pH of 5.0 and 50°C for 3 and 6 days with same amounts of total protein per g of cellulose using
  • Trichoderma reesei cellulolytic preparation Trichoderma reesei cellulolytic preparation, Thermoascus aurantiacus GH61 A polypeptide having cellulolytic enhancing activity (SEQ ID NO: 2 in WO 2005/074656), Aspergillus fumigatus beta-glucosidase (SEQ ID NO: 2 of WO 2005/047499), and Aspergillus aculeatus xylanase (Xyl I I disclosed in WO 94/21785) at dosage of 6 mg protein/g cellulose (cellulase 1 in Fig. 3);
  • Trichoderma reesei cellulolytic preparation with Aspergillus fumigatus CBH I and CBH II, Thermoascus aurantiacus GH61 polypeptide having cellulolytic enhancing activity SEQ ID NO: 2 in WO 2005/074656
  • Aspergillus fumigatus beta-glucosidase variant disclosed in co-pending US provisional application # 61/388,997 Aspergillus fumigatus xylanase (Xyl I II in WO 2006/078256) and Aspergillus fumigatus beta-xylosidase (disclosed in co-pending US provisional # 61/526833 or PCT/US12/052163 (Examples 16 and 17) at dosage of 6 mg protein/g cellulose (cellulase 3 in Fig. 3);
  • the sugar concentrations of samples diluted in 0.005 M H 2 S0 4 were measured using a 4.6 x 250 mm AMINEX® HPX-87P column (Bio-Rad Laboratories, Inc., Hercules, CA, USA) by elution with MilliQ-H 2 0 at 80°C at a flow rate of 0.6 ml per minute, and quantitation by integration of the glucose, cellobiose, and xylose signals from refractive index detection (CHEMSTATION®, AGILENT® 1 100 HPLC, Agilent Technologies, Santa Clara, CA, USA) calibrated by pure sugar samples. Cellulose conversion was calculated as % of theoretical based on initial cellulose and xylan input and concentrations of glucose and xylose obtained after enzyme hydrolysis.
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BR112014006853A BR112014006853A2 (pt) 2011-09-23 2012-09-21 composição de enzima, método para hidrolisar material celulósico acetilado, e, processo para produzir um produto de fermentação a partir de material celulósico acetilado
US14/343,002 US9714416B2 (en) 2011-09-23 2012-09-21 Cellulolytic enzyme compositions and uses thereof
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US14/279,559 US20140322766A1 (en) 2011-09-23 2014-05-16 C1-C2 Organic Acid Treatment of Lignocellulosic Biomass to Produce Acylated Cellulose Pulp, Hemicellulose, Lignin and Sugars and Fermentation of the Sugars
US14/279,550 US20140322763A1 (en) 2011-09-23 2014-05-16 C1-C2 Organic Acid Treatment of Lignocellulosic Biomass to Produce Acylated Cellulose Pulp, Hemicellulose, Lignin and Sugars and Fermentation of the Sugars
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WO2015156806A1 (en) * 2014-04-10 2015-10-15 Archer Daniels Midland Company Synthesis of r-glucosides, sugar alcohols, reduced sugar alcohols, and furan derivatives of reduced sugar alcohols
US20170121258A1 (en) * 2014-04-10 2017-05-04 Archer Daniels Midland Company Synthesis of r-glucosides, sugar alcohols, reduced sugar alcohols, and furan derivatives of reduced sugar alcohols
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